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This study evaluated the impact of a two-phage cocktail on an ex vivo colonic simulation model of gut microbiota derived from obese individuals, both in its normalized state and after enrichment with Enterobacter cloacae, an obesity-related bacteria. Microbiological analyses confirmed that the phage cocktail remained active throughout the colonic regions over three digestion cycles and effectively reduced enterobacterial populations in the enriched microbiota. Metabarcoding of the 16S rRNA gene revealed that phage therapy did not significantly alter the abundance of dominant genera, but selectively reduced E. cloacae across all colonic regions. Alpha diversity was significantly affected only in the enriched microbiota, while beta diversity analysis indicated significant compositional shifts during therapy, with reduced dispersion in the final treatment stage. Short-chain fatty acid profiling demonstrated region- and group-specific metabolic responses, with increased lactic and butyric acid concentrations in the ascending colon of the enriched microbiota following phage treatment. This study provides the first ex vivo evidence that a two-phage cocktail can selectively eliminate E. cloacae while preserving overall microbiota structure and functionality. These findings establish a foundation for future in vivo studies exploring the role of phage therapy in reshaping gut microbial communities and metabolic profiles, highlighting its potential as a precision tool for managing gut dysbiosis in metabolic disorders.<\/jats:p>","DOI":"10.3390\/ijms26062805","type":"journal-article","created":{"date-parts":[[2025,3,20]],"date-time":"2025-03-20T07:59:54Z","timestamp":1742457594000},"page":"2805","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["A Two-Phage Cocktail Modulates Gut Microbiota Composition and Metabolic Profiles in an Ex Vivo Colon Model"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-4549-4386","authenticated-orcid":false,"given":"Sthefhany Nohem\u00ed","family":"Rodr\u00edguez-Arellano","sequence":"first","affiliation":[{"name":"Facultad de Ciencias de la Nutrici\u00f3n y Gastronom\u00eda, Universidad Aut\u00f3noma de Sinaloa, Culiacan 80019, Sinaloa, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2994-4358","authenticated-orcid":false,"given":"Jean Pierre","family":"Gonz\u00e1lez-G\u00f3mez","sequence":"additional","affiliation":[{"name":"Laboratorio Nacional para la Investigaci\u00f3n en Inocuidad Alimentaria (LANIIA), Centro de Investigaci\u00f3n en Alimentaci\u00f3n y Desarrollo, A.C. 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